Decreased spontaneous electrical activity in neuronal networks exposed to radiofrequency 1800 MHz signals Corinne El Khoueiry, Daniela Moretti, Rémy Renom, Francesca Camera, Rosa Orlacchio, André Garenne, Florence Poulletier de Gannes, Emmanuelle Poque-Haro, Isabelle Lagroye, Bernard Veyret, et al. To cite this version: Corinne El Khoueiry, Daniela Moretti, Rémy Renom, Francesca Camera, Rosa Orlacchio, et al.. Decreased spontaneous electrical activity in neuronal networks exposed to radiofrequency 1800 MHz signals. Journal of Neurophysiology, American Physiological Society, 2018, 120 (6), pp.2719-2729. 10.1152/jn.00589.2017. hal-01943451 HAL Id: hal-01943451 https://hal.inria.fr/hal-01943451 Submitted on 17 Dec 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 DECREASED SPONTANEOUS ELECTRICAL ACTIVITY IN NEURONAL 2 NETWORKS EXPOSED TO RADIOFREQUENCY 1800 MHZ SIGNALS 3 4 Corinne El Khoueiry1, Daniela Moretti2, Rémy Renom1, Francesca Camera3, Rosa Orlacchio4, André 5 Garenne5, Florence Poulletier De Gannes1, Emmanuelle Poque-Haro1, Isabelle Lagroye1, 6, Bernard 6 Veyret1, 6 and Noëlle Lewis1 7 8 1 University of Bordeaux, CNRS, IMS, UMR 5218, Talence, France 9 2 Center of Synaptic Neuroscience and Technology, IIT, Genoa, Italy 10 3 La Sapienza University, DIET, Rome, Italy 11 4University of Rennes 1, IETR, Rennes, France 12 5 University of Bordeaux, CNRS, IMN, UMR 5293, Bordeaux, France 13 6 Paris “Sciences et Lettres” Research University, Paris, France 14 15 CORRESPONDENCE: 16 Noëlle Lewis 17 Email: [email protected] 18 IMS Laboratory, University of Bordeaux 19 351 Cours de la Libération 20 33405 Talence cedex, France EFFECT OF 1800 MHZ RF SIGNALS ON SPONTANEOUS ELECTRICAL ACTIVITY IN NEURONAL NETWORKS 21 ABSTRACT 22 So far, the only identified biological effects of radiofrequency fields (RF) are known to be caused by 23 heating but the issue of potential nonthermal biological effects, especially on the central nervous 24 system (CNS), remains open. We previously reported a decrease in the firing and bursting rates of 25 neuronal cultures exposed to a Global System for Mobile (GSM) RF field at 1800 MHz for 3 min 26 (Moretti et al. 2013). The aim of the present work was to assess the dose-response relationship for 27 this effect, and also identify a potential differential response elicited by pulse-modulated GSM and 28 continuous-wave (CW) RF fields. Spontaneous bursting activity of neuronal cultures from rat 29 embryonic cortices was recorded using 60-electrode Multi Electrode Arrays (MEAs). At 17-28 days in 30 vitro, the neuronal cultures were subjected to 15-min RF exposures, at SARs (Specific Absorption 31 Rates) ranging from 0.01 to 9.2 W/kg. Both GSM and CW signals elicited a clear decrease in bursting 32 rate during the RF exposure phase. This effect became more marked with increasing SAR and lasted 33 even beyond the end of exposure for the highest SAR levels. Moreover, the amplitude of the effect 34 was greater with the GSM signal. Altogether, our experimental findings provide evidence for dose- 35 dependent effects of RF signals on the bursting rate of neuronal cultures and suggest that part of the 36 mechanism is nonthermal. 37 38 Keywords: in vitro; neuronal cultures; radiofrequency fields; GSM-1800 signal; bursting rate. 39 40 NEW & NOTEWORTHY 41 In this study, we investigated the effects of some RF exposure parameters on the electrical activity of 42 neuronal cultures. We detected a clear decrease in bursting activity, dependent on exposure duration. 43 The amplitude of this effect increased with the SAR level and was greater with GSM than with CW 44 signal, at the same average SAR. Our experiment provides unique evidence of a decrease in 45 electrical activity of cortical neuronal cultures during RF exposure. 2 EFFECT OF 1800 MHZ RF SIGNALS ON SPONTANEOUS ELECTRICAL ACTIVITY IN NEURONAL NETWORKS 46 INTRODUCTION 47 The rapid development of wireless communications has raised questions about potential increased 48 health risks related to exposure to radiofrequency fields (RF). The close proximity between the mobile 49 phone and the brain of the user, combined with the fact that neurons are excitable cells, make the 50 central nervous system (CNS) a potential target of RF exposure. Absorption of RF fields by biological 51 tissues is quantified using the Specific Absorption Rate (SAR) metric, expressed in watts per 52 kilogram (W/kg), which represents the absorbed power per unit of tissue mass. Two types of RF 53 effects may occur: thermal or nonthermal. The former, which are prominent in case of high-level RF 54 exposures, are well established and understood, while the latter are still controversial (SCENIHR 55 2015). In this context, several human electroencephalography (EEG) studies have reported variations 56 in the EEG power spectrum during and/or after RF exposure, in resting EEG and during sleep (Van 57 Rongen et al. 2009; SCENIHR 2015), suggesting that RF exposure may directly influence brain 58 dynamics. However, the mechanisms underlying these effects on the EEG are still unknown. 59 Therefore, it was deemed necessary to study the spontaneous electrical activity of neuronal networks 60 in vitro, both at cellular and network levels, in order to detect effects of low-level RF on the nervous 61 system. 62 Our research group previously published an in vitro pilot study on neuronal networks exposed to a 63 Global System for Mobile (GSM) signal at 1800 MHz (Moretti et al. 2013). The GSM signal refers to 64 the initial development of digital mobile communication systems (2G). It is a TDMA (Time Division 65 Multiple Access) communication protocol. The pure carrier frequency emission is referred to as CW 66 (for Continuous Wave) while the GSM signal is a pulsed-modulated signal at 217 Hz with a 1/8 duty 67 cycle. In that previous study, a novel method was used to record neuronal extracellular electrical 68 activity under RF exposure. A 30% reversible decrease in firing and bursting rates was found during 69 3-min GSM 1800 MHz exposures of neuronal cultures from rat embryonic cortices (15-21 days in 70 vitro). The reported SAR of 3.2 W/kg was later recalibrated to 4.6 W/kg. To our knowledge, there has 71 been no new report on exposure in the GHz frequency range of neuronal networks since Moretti et al. 72 (2013). 73 The research presented here aimed to achieve more precise characterization of the previously 74 published effect, in terms of dose-effect and potential differential effect between pulsed (GSM) and 3 EFFECT OF 1800 MHZ RF SIGNALS ON SPONTANEOUS ELECTRICAL ACTIVITY IN NEURONAL NETWORKS 75 non-pulsed (CW) RF signals. This involved assessing the amplitude of the effect as a function of 76 SAR, electric field (E), and RF signal type (i.e. CW or GSM). The effects of all GSM and CW 77 exposures were compared at the same averaged SAR, so that the total energy transferred to the 78 sample was identical and the temperature elevation of the culture medium was the same. Data were 79 also plotted against the averaged E. The exposure protocol consisted of 15-min RF at bulk SARs 80 ranging from 0.01 to 9.2 W/kg, compared to sham conditions. 81 82 MATERIAL AND METHODS 83 Cell culture 84 To collect electrical activity, primary neurons were cultured on commercial microelectrode arrays 85 (MEAs) as described previously (Moretti et al. 2013). Polyethyleneimine (PEI) or polylysine (PLL) 86 (Sigma–Aldrich, St. Quentin-Fallavier, France) were used to coat the active area of the MEA to 87 promote attachment of neurons in the primary cell culture. These two coating methods have been 88 tested in order to spread over time the maturity of the neuron cultures, as cultures grown on PEI are 89 maturing sooner than neurons cultured on PLL. Laminin (Sigma–Aldrich, St. Quentin-Fallavier, 90 France) was also added on top of the PEI and PLL coatings for better adherence. 91 The primary neuronal cell cultures were obtained from the cortex of embryonic (E18) Sprague– 92 Dawley rats (Charles River Laboratories, L’Arbresle, France). After 5% isoflurane anesthesia, the 93 gestating rat was sacrificed by elongation. Embryos cortices were dissected in Dulbecco’s Modified 94 Eagle Medium (DMEM)-penicillin/streptomycin (Fisher Scientific, Illkirch, France), and treated for 30 95 min with an enzymatic solution containing 20 units/ml of papain and 0.005% of DNase (Worthington 96 Biochemical Corporation, Lakewood, Colorado, USA). The fragments were then subjected to 97 mechanical dissociation using a 10-ml serological pipette and centrifuged at 300 g for 5 min at room 98 temperature. The supernatant was eliminated and the pellet was placed in suspension in a solution 99 containing DNase. This latter mixture was placed above an albumin-inhibitor solution, to create a 100 discontinuous density gradient, and then centrifuged at 70 g for 6 min at room temperature. In this last 101 step, the dissociated cells (in the pellet) were separated from membrane fragments of dead cells in 102 the supernatant. Finally, the pellet containing cortical cells was suspended in the culture medium 4 EFFECT OF 1800 MHZ RF SIGNALS ON SPONTANEOUS ELECTRICAL ACTIVITY IN NEURONAL NETWORKS 103 composed of neurobasal medium supplemented with 2% B-27, 1% GlutaMAX, and 1% 104 penicillin/streptomycin (Fisher Scientific, Illkirch, France).